IA₃, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, Is Intrinsically Unstructured in Solution

Abstract

IA(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA(3) binds to YprA as an alpha-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113-117]. Surprisingly, only residues 2-32 of IA(3) are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.

Structural and Mechanistic Insights into the Interaction between Pyk2 and Paxillin LD Motifs

“…To further understand loop stabilization upon peptide binding, we also acquired steady-state 1 H- 15 N heteronuclear NOE measurements, which offer information on motions [49, 50] and can be used to identify order induced binding or ordered conformations in partially folded ensembles. Typically, the value for the heteronuclear 15 N [ 1 H] NOE of folded residues is ~1–0.7, and the NOE for a flexible loop is <0.5.…”

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling

“…Importantly, only residues 2-32 of IA 3 were seen in the X-ray structure, whereas the remaining residues are probably disordered in the complex . Recently, using CD and NMR spectroscopies it has been shown that IA 3 is unstructured in the absence of YprA (Green et al, 2004). The unfolded IA 3 samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K i ) of 1.7 nM.…”

“…The unfolded IA 3 samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K i ) of 1.7 nM. The addition of YprA led to a large spectral transition in IA 3 (Green et al, 2004). Thus, IA 3 is another example of an intrinsically disordered protein that folds when it recognizes its binding partner protein or that is stabilized in an otherwise transient conformation upon binding to its partner.…”

Key Features Determining the Specificity of Aspartic Proteinase Inhibition by the Helix-forming IA3 Polypeptide

“…By itself, free IA 3 is essentially unstructured (5), but, upon contact with its target proteinase, the polypeptide operates as an inhibitor through an unprecedented mechanism (3,4). Residues 2-32 become ordered and adopt an almost perfect amphipathic helical conformation occupying the active site of the enzyme (3,4).…”